In the last 10 to 15 years, two methods have prevailed in mass spectrometric analysis for soft ionization of biological macromolecules: Matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI). The biological macromolecules under analysis will be termed analyte molecules below. With the MALDI method, the analyte molecules are generally prepared on the surface of a sample support in a solid matrix, whereas with the ESI method they are dissolved in a liquid. Both methods have a considerable influence on the mass spectrometric analysis of biological macromolecules in genomics, proteomics and metabolomics; their inventors were awarded the Nobel prize for chemistry in 2002.
In a prepared MALDI sample, there are 103 to 105 times as many matrix molecules as there are analyte molecules, and they form a polycrystalline matrix in which the analyte molecules are integrated, either scattered in the interior of the crystals or at their grain boundaries. The prepared MALDI sample is briefly irradiated with a laser pulse, which is strongly absorbed by the matrix molecules. The pulsed irradiation causes the matrix to be explosively transferred from the solid aggregate state into the gaseous phase of a vaporization cloud (desorption). The analyte molecules are usually ionized by being protonated or deprotonated in reactions with matrix ions, the analyte ions being predominantly singly charged after leaving the vaporization cloud. The degree of ionization of the analyte molecules is only some 10−4. The term soft ionization is used when an analyte molecule is transferred in isolation into the gaseous phase and ionized, without suffering a dissociation.
Despite the linear absorption by the matrix, matrix-assisted laser desorption/ionization is a nonlinear process, which for pulsed laser radiation with a duration of a few nanoseconds only begins above an intensity threshold of around 106 watts per square centimeter. For soft ionization, the maximum intensity lies at an upper limit of approximately 107 watts per square centimeter. With a typical laser pulse duration of around ten nanoseconds, the stated intensity limits produce a fluence of between 10 and 100 millijoules per square centimeter.
The MALDI process is complex and affected by numerous factors, some of which are interdependent. Since the MALDI method was first published in 1988, many parameters have been investigated and varied. In spite of this, the processes in the matrix and in the vaporization cloud which lead to the ionization of the analyte molecules are still not completely understood and are still being intensely researched (K. Dreisewerd, Chem Rev. 103 (2003), 395-425: “The Desorption Process in MALDI”).
The chemical parameters of the MALDI process, for example the matrix substances themselves, the concentration ratios between matrix and analyte molecules and the preparation conditions, have been comprehensively researched. For analyte molecules of different chemical substance classes, such as proteins or nucleic acids, over one hundred different chemical matrix substances have been elucidated, such as sinapic acid, DHB (2,5-dihydroxy benzoic acid), CHCA (α-cyano-4-hydroxy cinnamic acid) or HPA (3-hydroxypicolinic acid). The matrix substances exhibit strong absorption in the wavelength range between 330 and 360 nanometers. A MALDI sample can be prepared in a number of different ways, for example with the “dried droplet” preparation or the “thin layer” preparation. In dried droplet preparation, the matrix substance is dissolved together with the analyte molecules in a solvent, applied to a sample support and then dried. In thin layer preparation, on the other hand, the matrix substance is applied to the sample support without analyte molecules and dried. A solution with analyte molecules is then applied to the thin polycrystalline matrix, causing the latter to be partially dissolved again, and the analyte molecules are integrated into the matrix during the subsequent drying. As far as the physical parameters of the MALDI process are concerned, it has, until now, been chiefly the temporal duration of the laser pulses, the intensity in the laser focus and the wavelength of the pulsed laser beam which have been considered.
For commercially available mass spectrometers with MALDI, pulsed laser systems in the ultraviolet spectral range (UV) are predominantly used nowadays. A number of laser types and wavelengths are available: nitrogen lasers (λ=337 nm), excimer lasers (λ=193 nm, 248 nm, 308 nm) and Nd:YAG lasers (λ=266 nm, 355 nm). Only the nitrogen laser and the Nd:YAG laser at a wavelength of 355 nanometers are of interest commercially for the MALDI method, and the nitrogen laser is far and away the one most frequently used. The laser medium of the nitrogen laser and the excimer laser is a gas or a gas mixture, whereas with the Nd:YAG laser it is a YAG (yttrium aluminium garnet: Y3Al5O12) crystal doped with neodymium ions. With the Nd:YAG laser, the strongest laser line, at a wavelength of 1,064 nanometers, is transformed into the stated wavelengths in nonlinear optical crystals. The duration of the laser pulses used in the MALDI method is typically between 1 and 20 nanoseconds in the UV; in the academic field, however, pulse durations in the region of picoseconds have also been used.
For the MALDI method, laser systems which emit in the infrared spectral region (IR): Er:YAG (λ=2.94 μm) and CO2 (λ=10.6 μm) are also occasionally used for research purposes. Whereas with the UV-MALDI method the matrix molecules are supplied with energy via excited electronic states, in the IR-MALDI method it is oscillations of the matrix molecules that are excited. The pulse duration of the IR laser systems in the IR-MALDI method are between 6 and 200 nanoseconds. In contrast to the UV-MALDI method, both solid matrices and liquid matrices, for example glycerine, are used in the IR-MALDI method.
A laser system usually comprises a laser medium, an energy input for the excitation of the laser, an optical resonator and optical and electrooptical components to shape the laser beam. In the following, a laser system is understood to be the complete set-up comprising optical, electrical and electrooptical components which are necessary to generate and shape the laser beam from the laser medium to the location of the MALDI sample. The components for beam formation can be located both inside the optical resonator, in the vicinity of the laser medium and also outside the optical resonator. These types of components include lenses, mirrors, active and passive Q-switches for pulse generation, coupling and decoupling into and out of an optical fiber, and nonlinear optical crystals. To those skilled in the art it is apparent that not all the components mentioned have to be used in the various laser systems, and that they can be supplemented by further components.
The laser systems used in the MALDI method differ not only in their wavelength but also in their spatial beam profile. For solid-state lasers such as the Nd:YAG laser or the Er:YAG laser, the laser medium is a crystal doped with ions. The laser medium is located in an optical resonator, which ensures that the spatial beam profile consists of a transverse basic mode or a few transverse modes. The radial intensity distribution of the transverse basic mode corresponds to a Gaussian function and is rotationally symmetric to the direction of propagation of the laser beam. A laser beam like this can be focused to a minimum diameter which is limited only by the diffraction.
The nitrogen laser at a wavelength of 337 nanometers is far and away the most frequently used type of laser in the MALDI method, this wavelength being the most intensive laser line of the nitrogen laser. The laser medium used is gaseous nitrogen, which is excited by means of an electrical discharge between two electrodes. Since the most intensive laser line exhibits a high amplification, a laser pulse can decrease the population inversion of the energy states even if it sweeps the electrodes only once. Even when using cavity mirrors, many transverse modes are superimposed in the beam profile of the nitrogen laser, with the result that the minimum diameter of a laser focus in commercial nitrogen lasers at a wavelength of 337 nanometers is only around three micrometers. The typical diameter of the area irradiated in MALDI applications is around 20 to 200 micrometers. The beam profile of the nitrogen laser has an almost flat top at the electrodes, the width and height of the beam profile being determined by the separation or the height of the discharge electrodes. The repetition rate of the laser pulses in the nitrogen laser is limited to around 100 hertz unless provision is made for a rapid gas exchange. Nitrogen lasers with a typical repetition rate of 50 hertz are used for MALDI applications.
In practice, the electrical gas discharge in the nitrogen laser is not the same at every point between the electrodes, and it generates a spatially inhomogeneous amplification profile which does not even out during the short time the laser is in action, but instead transfers to the beam profile of the nitrogen laser. The nitrogen laser thus has a spatially modulated flat-top beam profile with intensity maxima and minima which is imaged onto the sample or focused onto it. Furthermore, a pulsed gas discharge is normally difficult to reproduce, so that the intensity distribution in the beam profile and the energy fluctuate from laser pulse to laser pulse. According to the prior art, the MALDI method strives for an intensity distribution with maximum spatial homogeneity on the sample. If this is generated over a wide area, the inhomogeneities of the prepared MALDI sample, as occur, for example, with an uneven distribution of the analyte molecules in the matrix, are averaged out. The immanently present inhomogeneities in the beam profile of the nitrogen laser do, however, lead to the intensity distribution on the sample being spatially modulated and exhibiting undesirable intensity maxima.
The pulsed solid-state lasers used until now in the MALDI method typically have a beam profile which comes very close to a single Gaussian mode. If a pulsed laser beam is focused or imaged onto the sample, then there is a Gaussian intensity distribution with a single maximum at the location of the sample. With solid-state lasers in the UV, the half-width, at which the intensity of the maximum has fallen off to half the value, can theoretically be less than one micrometer, but in MALDI applications it is around 20 to 200 micrometers. Even if laser pulse repetition rates of several hundred kilohertz are possible in principle with solid-state lasers, most current MALDI applications work with a repetition rate of up to 200 hertz. The energy fluctuations from laser pulse to laser pulse are typically less in the case of solid-state lasers than with nitrogen lasers.
According to the prior art, the objective is a spatially homogeneous intensity distribution on the sample. In order to obtain a homogeneous intensity distribution on the sample with the Gaussian beam profile of a solid-state laser, the beam profile can be spatially homogenized by propagation in an optical fiber and then imaged onto the sample. To facilitate this, the laser beam is coupled into an optical fiber in which a plurality of transverse fiber modes with differing radial intensity distributions can propagate (multimode fibers). The propagation of the coupled laser beam in the multimode fiber means that energy is transferred out of the Gaussian mode into a plurality of transverse fiber modes which are superimposed at the output of the optical fiber. If the temporal coherence of the laser beam used is sufficiently low or the multimode fiber is sufficiently long, the intensity distribution at the fiber output is given by the sum of the intensity distributions of the individual transverse fiber modes. The plurality of transverse fiber modes with differing radial intensity profiles thus results in a homogeneous intensity distribution at the fiber output. If the output of the multimode fiber is now imaged, a flat-top intensity distribution is also obtained on the sample. This method of homogenizing the beam profile can also be used with the nitrogen laser to minimize the immanent inhomogeneities in the beam profile.
With some MALDI mass spectrometers the laser beam is imaged or focused onto the sample through a metal grid, which acts as an ion focusing system with which the ions are drawn out of the vaporization cloud. Since this ion focusing system must permit a high transmission rate of the ions generated in the MALDI process, the open areas of the metal grid are so large, compared with the bar area, that they have little effect on the homogeneous intensity distribution on the sample.
The quality of a mass spectrometric analysis is generally determined by the following parameters: The mass accuracy, the mass resolution, the detection power, the quantitative reproducibility and the signal-to-noise ratio. This means that the quality of a mass spectrometric analysis increases if at least one parameter is improved and the other parameters do not deteriorate as a result. The mass accuracy includes both a systematic deviation of the measured average ion mass from the true ion mass (mass trueness or rather the deviation from mass trueness) as well as the statistical variance of the individual measured values around the mean of the ion mass (mass precision). The mass resolution determines which ion masses in the mass spectrometric analysis can still be distinguished. In practice, however, it is not only the quality but also the robustness of the mass spectrometric analysis that is important. A mass spectrometric analysis is robust if its quality changes little when the measuring parameters, for example the energy of the laser pulses or the preparation conditions of the MALDI sample, are varied.
The ion signal of a mass spectrometer with MALDI is proportional to the ionization efficiency, to the desorbed sample volume and to the concentration of the analyte molecules in the sample. The ionization efficiency is given by the number of analyte ions which can be evaluated, divided by the number of analyte molecules in the desorbed sample volume, i.e. the percentage of the analyte molecules from the sample volume removed by the laser irradiation which is available as ions for a mass spectrometric analysis. If analyte molecules are already present in the matrix in an ionized form before the desorption process, the number of analyte molecules is increased by the number of analyte ions present which are already ionized. Since the desorbed sample volume can be relatively easily increased by the irradiated sample area and by the fluence, the ionization efficiency represents an important parameter for the optimization of the MALDI process. A high ionization efficiency facilitates a high detection power because a maximum ion signal at low concentration (or low sample utilization) is achieved. With a typical degree of ionization of only 10−4 it is possible to considerably improve the MALDI process. The definition of the ionization efficiency of the MALDI process also takes into account the losses which arise as a result of a fragmentation of analyte molecules during the transfer into the gaseous phase and which therefore reduce the number of analyte ions which can be evaluated.
For the mass spectrometric analysis of the analyte ions generated in the MALDI process, both conventional sector field mass spectrometers and quadrupole mass spectrometers as well as quadrupole ion trap mass spectrometers and ion cyclotron resonance mass spectrometers are possible in principle. Particularly suitable, however, are time-of-flight mass spectrometers with axial injection, which require a pulsed current of ions to measure the time of flight. In this case, the starting time for the time of flight measurement is predetermined by the ionizing laser pulse. The MALDI process was originally developed for use in a vacuum. In more recent developments, matrix-assisted laser desorption/ionization is also used at atmospheric pressure (AP MALDI). Here, the ions are generated with a repetition rate of up to 2 kilohertz and fed, with the help of an ion guide, to a time-of-flight mass spectrometer with orthogonal ion injection (abbreviated OTOF), a quadrupole ion trap mass spectrometer or an ion cyclotron resonance mass spectrometer. In an OTOF mass spectrometer, the ions generated in the MALDI process can be fragmented and stored before the measurement of the time of flight with an electronic pulsed injection is commenced.
With specific MALDI applications, the intensity on the sample is increased to such a degree that the ions generated have enough intrinsic energy to dissociate. Depending on the time between the generation of the ions and their dissociation, this is termed a decay within the ion source (ISD or “in source decay”) or outside the ion source (PSD or “post source decay”).
Moreover, there are also imaging mass spectrometric analytical methods (IMS or “imaging mass spectrometry”), in which the MALDI process is used to generate the ions. With IMS, a thin section of tissue obtained, for example, from a human organ using a microtome, is prepared with a matrix substance and mass spectrometrically analyzed after being spatially resolved. The spatial resolution of the mass spectrometric analysis can be done either by scanning individual spots of the tissue section or by stigmatic imaging of the ions generated. With the scanning method, the pulsed laser beam is focused onto a small diameter on the sample, and a mass spectrum is measured for each individual pixel. A one- or two-dimensional frequency distribution is produced for individual proteins from the plurality of individual spatially-resolved mass spectra. With stigmatic imaging, an area of up to 200 by 200 micrometers is irradiated homogeneously with a laser pulse. The ions generated in this way are imaged ion-optically pixel by pixel onto a spatially-resolving detector. Until now it has only been possible to scan the frequency distribution of one ion mass with a single laser pulse because spatially resolving ion detectors that operate fast enough are not available. The measured ion mass can be varied from laser pulse to laser pulse, however.